Carboxylated derivatives of glycosaminoglycans and use as drugs

ABSTRACT

A glycosaminoglycan derivative endowed with heparanase inhibitory activity and antitumor activity, bearing carboxylate groups in positions 2 and 3 of at least part of the glycosaminoglycan residues, and to the process for preparing the same. The glycosaminoglycan derivatives of the present invention are generated starting from natural or synthetic glycosaminoglycans, preferably heparin or low molecular weight heparin, optionally 2-O- and 2-N-desulfated by two steps of oxidation. By the first oxidation, adjacent dials and optionally adjacent OH/NH2 of the glycosaminoglycan residues are converted to aldehydes and by the second oxidation said dialdehydes are converted to carboxylate groups. The first oxidation preferably leads to the cleavage of C2-C3 linkage of the ring of oxidable residues. The invention relates to a process for the preparation of said glycosaminoglycan derivatives and to their use as active ingredients of medicaments.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This patent application is a continuation of co-pending U.S. patentapplication Ser. No. 15/034,555, filed May 5, 2016, which is a NationalStage Entry of PCT/EP2014/072676, filed Oct. 22, 2014, which claimspriority to Italian Application No. LO2013A000006, filed Nov. 6, 2013,the entire teachings and disclosure of which are incorporated herein byreference thereto.

BRIEF SUMMARY OF THE INVENTION

The invention relates to glycosaminoglycan derivatives, endowed withheparanase inhibitory activity and antitumor activity, bearingcarboxylate groups in positions 2 and 3 of at least part of theglycosaminoglycan residues, and to the process for preparing the same.

The glycosaminoglycan derivatives of the present invention are generatedstarting from natural or synthetic glycosaminoglycans, optionallymodified chemically or enzymatically. In particular, saidglycosaminoglycan derivatives are obtainable by two steps of oxidationof the glycosaminoglycan. By the first oxidation (a), preferably byperiodate, adjacent diols and optionally adjacent OH/NH₂ of theglycosaminoglycan residues are converted to aldehydes and by the secondoxidation (b) said dialdehydes are converted to carboxylate groups.According to the present invention, preferably from 10% to 100%, morepreferably from 25% to 100%, of 2-O-non-sulfated residues and optionally2N-, 3-O-non sulfated residues of a glycosaminoglycan are oxidized inthe first oxidation step (a), under conditions effective to convertadjacent diols and optionally adjacent OH/NH₂ to aldehydes.

The first oxidation preferably leads to the cleavage of C₂-C₃ linkage ofthe ring of oxidable residues. Preferably, the glycosaminoglycan is anatural or synthetic glycosaminoglycan, optionally 2-O- and/or2-N-desulfated, having a sulfation degree (SO₃—/COO-molar ratio) of from0.8 to 2.8, preferably of from 0.9 to 2.5. The sulfation degree(SO₃—/COO— molar ratio) is herein intended as determined byconductimetric titration according to Casu B. and Gennaro U., 1975,Carbohydr Res 39, 168-176. The di/tricarboxylated glycosaminoglycanderivatives obtainable by the inventive process disclosed above show acarboxyl increment of from 1.2 to 2.2, wherein the carboxyl increment iscalculated as the ratio of the sulfation degree (SO₃—/COO— molar ratio)of the starting material to the sulfation degree (SO₃—/COO— molar ratio)of the di/tricarboxylated derivative, determined by conductimetrictitration as herein defined. More specifically, the sulfation degree ofthe starting glycosaminoglycan is determined on a sample of theglycosaminoglycan intermediate obtained by the first oxidation step (a),after reduction by NaBH₄.

Preferably, the glycosaminoglycan derivatives of the present inventionare obtained starting from natural heparins from any animal and organsource, or from synthetic heparins, optionally chemically orenzymatically modified. More preferably, the starting material isunfractionated heparin, low-molecular-weight heparins (LMWHs, which havea molecular weight of from 3,500 to 8,000 Da.), heparan sulfate (HS) orderivatives thereof. Most preferred are glycosaminoglycan derivativesobtainable from unfractionated heparins or from LMWHs, optionally 2-O-and/or 2-N-desulfated.

The invention further relates to a process for the preparation of saidglycosaminoglycan derivatives and further to their use as activeingredients of medicaments, optionally in combination with knownestablished drugs or treatments. In particular, the present invention isdirected to said glycosaminoglycan derivatives for use as medicamentsfor the treatment of pathological conditions, such as multiple myelomaand other neoplasias (i.e. sarcomas, carcinomas, hematologicalmalignancies), including their metastatic forms.

Furthermore, the invention relates to the use of said glycosaminoglycanderivatives in any therapeutic indication gaining benefit from theinhibition of heparanase (i.e. diabetic nephropathy, inflammatory boweldisease, colitis, arthritis, psoriasis, sepsis, atherosclerosis). Theinvention also relates to pharmaceutical compositions comprising saiddi/tricarboxylated glycosaminoglycan derivatives, in particular topharmaceutical compositions comprising di/tricarboxylated heparin andlow molecular weight heparin (LMWH) derivatives, as active agent.Optionally, said pharmaceutical compositions further comprise at leastone different active agent, preferably at least one differentantitumoral agent.

BACKGROUND OF THE INVENTION

Multiple myeloma is the second most prevalent hematologic malignancy andaccounts for over 10% of all hematologic cancer in Unites States, witharound 20,000 new cases each year and a mortality greater than 50%(Graham-Rowe D., 2011, Multiple myeloma outlook. Nature 480, s34-s35).

Over the last few years, promising therapies have been developed, suchas the administration of proteasome inhibitor (Velcade), bisphosphonatesand thalidomide among others. The effectiveness of these agents is, atleast in part, due to their impact on the myeloma tumormicroenvironment.

Although efficacy of said agents against myeloma has been demonstrated,the need remains for new and improved drugs for treating myeloma andother tumors.

Heparanase is an endo-13-glucuronidase that cleaves heparan sulfate (HS)of HS-proteoglycans, such as syndecan-1, thereby releasing HS-boundgrowth factors.

In humans, there appears to be a single dominant functional heparanaseenzyme capable of cleaving HS. Heparanase is expressed in most humantumors, where it significantly increases both the angiogenic and themetastatic potential of tumor cells. Elevated heparanase levels havebeen in fact correlated with advanced progression and metastasis of manytumor types. For example, high level of heparanase is associated with ashorter post-operative survival time of patients. A direct role ofheparanase in tumor metastasis has been demonstrated in Vlodaysky's andSanderson's laboratory, where our novel inhibitors have been tested.

In addition to its enzymatic functions, that include release of HS-boundgrowth factors and degradation of the extracellular matrix (ECM),heparanase has also a non-enzymatic function that may impact tumorbehavior and microenvironment. Sanderson's group pioneered the study ofheparanase and syndecan-1 in myeloma, establishing that heparanase actsas a master regulator of aggressive tumor phenotype in myeloma. Thisoccurs by promoting the up-regulation of VEGF and MMP-9, that togetherstimulate tumor growth, metastatic and osteolytic bone destruction. Itwas in fact demonstrated in vivo that heparanase promotes the growth ofmyeloma tumors and spontaneous metastasis to bone and that heparanaseexpression by tumor cells fuels rampant osteolysis, at least partiallydue to up-regulation of RANKL expression. The osteolysis promotingeffect of heparanase may be of great importance because bone-boundgrowth factors are released when bone is degraded. In addition,osteoclasts can release tumor growth promoting factors such as HGF.Together these factors may help establish niches within the bone marrowthat support tumor cell homing and subsequent growth (Fux, L. et al.,2009, “Heparanase: busy at the cell surface.” Trends Biochem Sci, 34(10): 511-519; Sanderson R. D. and Yang Y., 2008, “Syndecan-1: a dynamicregulator of the myeloma microenvironment.” Clin Exp Metastasis25:149-59; Ilan N. et al., 2006, “Regulation, function and clinicalsignificance of heparanase in cancer metastasis and angiogenesis.” Int JBiochem Cell Biol, 38: 2018-2039). Inhibition of heparanase is thus afeasible target of myeloma therapy, supported by the fact that there isa single enzymatically active heparanase in humans and by the fact thatits expression in normal tissues is rare. Furthermore, it has been shownthat heparanase knock-out mice are viable and exhibit no visibledisorders. This indicates that little or no side effect can derive froma heparanase inhibition strategy (Casu B., et al. 2008.Non-anticoagulant heparins and inhibition of cancer. PathophysiolHaemost Thromb. 36: 195-203; Vlodaysky I., et al. 2007. Heparanase:structure, biological functions, and inhibition by heparin-derivedmimetics of heparan sulfate. Curr Pharm Des. 13: 2057-2073; Naggi A., etal. 2005. Modulation of the Heparanase-inhibiting Activity of Heparinthrough Selective Desulfation, Graded N-Acetylation, and GlycolSplitting. J. Biol. Chem. 280: 12103-12113).

Heparin is a linear polydisperse sulfated polysaccharide of theglycosaminoglycan family, endowed with anticoagulant and antithromboticactivity. The saccharidic chains of heparin consist of alternatinguronic acid and D-glucosamine. The major repeating unit is 2-O-sulfatedL-iduronic acid (IdoA2S)a(1-4) and N-, 6-O-disulfated D-glucosamine(GlcN6S). Minor constituents are non-sulfated L-iduronic andD-glucuronic acid, along with N-acetyl D-glucosamine and N-, 3-O-,6-O-trisulfated D-glucosamine (Casu B., 2005. “Structure and activedomains of heparin.” In: Chemistry and biology of heparin and heparansulfate. Amsterdam: Elsevier. 1-28; Casu B. and Lindahl U., 2001,“Structure and biological interactions of heparin and heparan sulfate.”Adv Carbohydr Chem Biochem, 57: 159-206). Heparin, which is structurallysimilar to HS, can efficiently inhibit heparanase, but its use ofheparin at high doses in a heparanase inhibition strategy is howeverimpossible due to its anticoagulant activity.

Interestingly, low molecular weight heparins (LMWHs), which are morebioavailable and less anticoagulant than heparin, appear to prolongsurvival of patients with cancer, probably through direct effect ontumor growth and metastasis. This may be due, at least in part, toinhibition of heparanase enzyme activity (Zacharski L. R., and Lee, A.Y. 2008. Heparin as an anticancer therapeutic. Expert Opin InvestigDrugs 17:1029-1037).

Effective inhibitors of the enzymatic activity of heparanase have beenselected in the prior art by studying heparanase inhibition bynon-anticoagulant heparins, most of which contain non-sulfated uronicacid residues modified by opening of the glucosidic ring by cleavage ofthe linkage between carbons 2 and 3 of a glycosaminoglycan residue(glycol-splitting). Said inhibitors differ in their 0-sulfationpatterns, degree of N-acetylation and glycol-splitting of bothpre-existing non-sulfated uronic acid residues and those generated bygraded 2-O-desulfation (Naggi A., 2005. “Glycol-splitting as a devicefor modulating inhibition of growth factors and heparanase inhibition byheparin and heparin derivative.” In: Chemistry and Biology of Heparinand Heparan Sulfate. Amsterdam: Elsevier 461-481; Yang Y. et al., 2007,“The syndecan-1 heparan sulfate proteoglycan is a viable target formyeloma therapy.” Blood, 110:2041-2048).

In particular, the terms “glycol split” (gs) conventionally refers topolysaccharides that present opening of some monosaccharide residues dueto the break (glycol splitting) of one linkage between two adjacentcarbons, each bearing an hydroxyl group. A first generation glycol splitheparins, i.e. the so-called “reduced oxyheparins” (RO-heparins),consisted of unmodified polysulfated blocks occasionally interrupted byglycol split residues, corresponding to non-sulphated glucuronicacid/iduronic acid residues that were present along the original chain,first oxidized to dialdehydes and then reduced to alcohols (Naggi A.,2005, “Glycol-splitting as a device for modulating inhibition of growthfactors and heparanase inhibition by heparin and heparin derivative.”In: Chemistry and Biology of Heparin and Heparan Sulfate. Amsterdam:Elsevier 461-481).

WO 01/55221 discloses glycosaminoglycans with a 2-O-desulfation degreenot greater than 60% of the total uronic acid units. Saidglycosaminoglycans are devoid of anticoagulant activity and showantiangiogenic activity based on the inhibition of FGFs. No activity wasforeseen for inhibition of heparanase.

US 2008/0051567 discloses a compound corresponding to a 100%N-acetylated and 25% glycol split heparin, exerting little or noanticoagulant activity or release of growth factors from theextracellular matrix. Said compound was found to inhibit heparanase,tumor growth, angiogenesis and inflammation in experimental animalmodels, including Sanderson's model of myeloma (Yang Y., et al. 2007.The syndecan-1 heparan sulfate proteoglycan is a viable target formyeloma therapy. Blood 110:2041-2048).

Despite that, the need remains for providing improved compounds withhigher heparanase affinity, higher selectivity and improvedbioavailability, being suitable for treating heparanase-relatedpathologies, such as myeloma and other tumors.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: prevalent structures generated by the process of the presentinvention. (1) disaccharidic unit of a glycosaminoglycan polymercomprising one uronic acid (iduronic and/or glucuronic) and oneglucosamine (2-N-acetylated, 2-N-unsubstituted and/or 2-N-sulfated), inwhich the hydroxyl groups (R₃ and R₄) can each or both be substituted bya sulphate group or non-substituted. (2, 3, 4) representative structures(new entities) generated by oxidative cleavage of the ring of2-non-sulfated heparin residues, followed by further oxidation to tri-or di-carboxylated residues.

FIG. 2: one example of chromatogram obtained with MS detection. Thepresence of peaks corresponding to oligosaccharides containing theuronic acid di- or tri-carboxylate residues, attributed by the measuredvalues of molecular weight, are highlighted respectively by thecode“+carbox2” or “+carbox3” in the graph.

FIG. 3: one example of chromatogram obtained with MS detection. Thepresence of peaks corresponding to oligosaccharides containing theuronic acid di- or tri-carboxylate residues, attributed by the measuredvalues of molecular weight, are highlighted respectively by thecode“+carbox2” or “+carbox3” in the graph.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a novel class of chemically modifiedglycosaminoglycan derivatives, endowed with heparanase inhibitoryactivity. In particular it relates to carboxylated glycosaminoglycanderivatives, wherein at least part of the residues are split residuesbearing three carboxylate groups (or two, if the split residue is aglucosamine).

The glycosaminoglycan derivatives of the present invention arepreferably heparin derivatives, designed as “di/tricarboxylatedheparins”, which strongly inhibit the heparan sulfate degrading activityof heparanase. The chemical modifications made to heparin, modifying theresidue of glucuronic acid included in the binding site for ATIII,abolish the heparin anticoagulant activity, enabling the use of highdoses.

The glycosaminoglycan derivatives of the present invention areobtainable by oxidation, preferably by periodate, of 2-non-sulfatedresidues of a glycosaminoglycan under conditions effective to convertadjacent diols and optionally adjacent OH/NH₂ of the glycosaminoglycanto aldehydes, followed by oxidation of the oxidized glycosaminoglycanunder conditions to convert said dialdehydes to carboxylate groups. Inparticular, the glycosaminoglycan derivatives of the present inventionare preferably obtainable starting from a glycosaminoglycan having asulfation degree (SO₃—/COO— molar ratio) of from 0.8 to 2.8, preferablyof from 0.9 to 2.5. The sulfation degree (SO₃—/COO— molar ratio) isherein intended as determined by conductimetric titration according toCasu B. and Gennaro U., 1975, Carbohydr Res 39, 168-176. Thedi/tricarboxylated glycosaminoglycan derivatives obtainable by theinventive process disclosed above show a carboxyl increment of from 1.2to 2.2, wherein said carboxyl increment is calculated as the ratio ofthe sulfation degree (SO₃—/COO— molar ratio) of the starting material,after the first oxidation step, to the sulfation degree (SO₃—/COO— molarratio) of the di/tricarboxylated derivative, determined byconductimetric titration as herein defined. More specifically, thesulfation degree of the starting material after the first oxidation stepis determined on a sample of the glycosaminoglycan intermediate obtainedby the first oxidation step (a), after reduction by NaBH₄.

Preferably, the glycosaminoglycan derivatives of the present inventionderive from natural or synthetic (chemically or enzymatically obtained)glycosaminoglycans, more preferably from 2-O- and/or 2-N-desulfatedglycosaminoglycans. In a preferred embodiment, said natural or syntheticglycosaminoglycan is unfractionated heparin, LMWH, or heparan sulfate,optionally 2-O- and/or 2-N-desulfated.

More preferably the glycosaminoglycan derivatives derive from natural orsynthetic heparins or LMWHs, 2-O- and optionally 2-N-desulfated.

As an example, heparin chains can naturally comprise from about 5% to35% of 2-O-non-sulfated uronic acid residues, from 0% to 50% ofN-acetylated glucosamine residues and from about 0% to 6% ofN-unsubstituted (neither N-sulfated, nor N-acetylated) glucosamineresidues. Different sulfation degree depends on the heparin source(animal species, organ sources) and on the extraction procedures. Every2-O- or 2-N-non-sulfated residue of glycosaminoglycans, not bearing3-0-sulfate substituents, is susceptible of oxidation with ring opening(split) and conversion of vicinal diols and OH/NH₂ to aldehydes.Optionally, graded 2-O-desulfation of the starting glycosaminoglycansallows to modulate the ratio of glucosamine/uronic acid split residues.

The invention further relates to the process for preparing saidcarboxylated glycosaminoglycan derivatives and further to their use asactive ingredients of medicaments for treating pathological conditions,either as sole active ingredient or in combination with othermedicaments. Said pathological conditions comprise multiple myeloma andother neoplastic diseases, including their metastatic forms.Furthermore, the invention relates to said carboxylatedglycosaminoglycan derivatives for use in any therapeutic indicationgaining benefit from inhibition of heparanase. The invention alsorelates to pharmaceutical compositions containing said carboxylatedglycosaminoglycan derivatives, optionally in combination with at leastone further active ingredient.

The process for preparing the carboxylated glycosaminoglycan derivativesof the invention comprises: oxidation, preferably by periodate (or byuse of oxidizing reagents having similar reactivity), of the susceptiblenon-sulfated residues of a glycosaminoglycan, under conditions effectiveto convert adjacent diols and optionally adjacent OH/NH₂ to aldehydes,followed by oxidation of the glycosaminoglycan resulting from the firstoxidation, under conditions to obtain two new carboxylate groups fromthe corresponding aldehyde groups.

The process of the present invention thus comprises the oxidation,preferably by periodate, of 10% to 100%, preferably of from 25% to 100%,of 2-O- and optionally 2-N-, 3-O-non-sulfated residues of aglycosaminoglycan, under conditions effective to convert adjacent diolsand optionally adjacent OH/NH₂ to aldehydes; then it comprises theoxidation of the oxidized glycosaminoglycan under conditions effectiveto convert said dialdehydes to carboxylate groups.

Preferably, the starting glycosaminoglycan is a natural or syntheticglycosaminoglycan; more preferably it is selected from heparin, lowmolecular weight heparins, or heparan sulfate; most preferably it isselected from heparin and LMWHs, 2-O- and optionally 2-N-desulfated.

Preferably, the starting glycosaminoglycan has a sulfation degree(SO₃—/COO—) of from 0.8 to 2.8, more preferably of from 0.9 to 2.5, byconductimetric titration, as defined herein.

The first oxidation of the inventive process is preferably carried outat a pH of from 3 to 10, more preferably at a pH of from 4.5 to 8. In apreferred embodiment the first oxidation of the inventive process iscarried out at a pH of from 3 to 5, in order to cleave only the linkageC₂-C₃ of the non-sulfated uronic acid residues, avoiding side reactions.In another preferred embodiment, the first oxidation of the inventiveprocess is carried out at a pH of from 5.5 to 10, in order to cleave thelinkage C₂-C₃ of both the 2-O-non-sulfated uronic acid andN-non-sulfated glucosamine residues.

In a preferred embodiment, the first oxidation is performed underconditions to cleave the bond between the C2 and C3 of both the2-O-non-sulfated uronic acids and of the 2-N-, 3-O-non-sulfatedglucosamines.

Optionally, the inventive process is performed in the presence of NTA(nitrilotriacetic acid), a chelating and sequestering agent used toreduce depolymerization, in the presence of NaHCO₃ or pyridine, toalkalinize the reaction solution, or in the presence of MnCl₂ with orwithout NTA. The further dialdehyde oxidation is preferentiallyperformed using NaClO₂, or by the use of agents with comparableoxidizing properties, such as TEMPO (2,2,6,6 TetraMethyl-1-Piperidinyl-Oxy).

Preferably, carboxylated uronic acid residues in the glycosaminoglycansderivatives of the present invention are from 25% to 100%, morepreferably from 50% to 100%, most preferably from 60% to 90%, of thetotal residues of the total carboxylated residues of theglycosaminoglycan.

The carboxylated glycosaminoglycan derivatives obtainable by the aboveprocesses preferably have a molecular weight of from 8000 to 30,000 Da.,depending on the process conditions and on the startingglycosaminoglycan employed. In a preferred embodiment, more preferablywhen unfractionated heparin is employed as the startingglycosaminoglycan, the carboxylated glycosaminoglycan derivativeobtainable by the above processes preferably has a molecular weight offrom 3,000 to 20,000 Da., preferably from 3,500 to 12,000 Da.

The novel glycosaminoglycan derivatives obtainable by the process of thepresent invention represent a new class of heparin-like polysaccharides,characterized by the presence of split residues, each bearing twoadditional carboxylate groups. Note that residues bearing one naturalcarboxylate group, are converted to tricarboxylated residues by theprocess of the present invention. Said novel di/tricarboxylatedglycosaminoglycan derivatives have unexpectedly shown to be strongheparanase inhibitors in vitro and to inhibit myeloma in animal models.

Glycosaminoglycan derivatives comprising residues bearing two or threecarboxylate groups, being also less sulfated than the parentglycosaminoglycan, display more favorable pharmacokinetics than theiranalogues bearing less carboxylic groups.

The present invention further relates to the compounds obtainable by theabove processes for use as medicaments.

In particular, the present invention relates to the compounds obtainableby the above processes for use as antitumor, preferably for use asantimyeloma medicaments, either alone or in combination with at leastone further active ingredient.

Heparin and low molecular weight heparin derivatives prepared accordingto the present invention have shown effective inhibition of heparanaseactivity, both in vitro and in vivo in a multiple myeloma experimentalmodel.

Examples

Compounds Preparation

Samples of unfractionated or fractioned heparins, endowed with differentdegree of sulfation (SO₃ ⁻/COO⁻) by conductimetric titration, weresubjected to periodate oxidation (to give split dialdehyde units),performed by modification of known methods. Graded 2-O-desulfation ofunfractionated heparins (UFH) was performed following modification ofknown methods (Jaseja M. et al., 1989, “Novel regio- andstereo-selective modifications of heparin in alkaline solution. Nuclearmagnetic resonance spectroscopic evidence.” Canad J Chem, 67, 1449-1455;R. N. Rej and A. S. Perlin, 1990, “Base-catalyzed conversion of thea-L-iduronic acid 2-sulfate unit of heparin into a unit ofa-L-galacturonic acid and related reactions.” Carbohydr. Res. 200, 25,437-447; Casu B. et al., 2004, “Undersulfated and Glycol-Split HeparinsEndowed with Antiangiogenic Activity.” J. Med. Chem., 47, 838-848).Under basic conditions, 2-O-desulfated (natural or chemically induced)L-iduronic acid units are converted into 2,3-epoxy derivatives andfinally to L-galacturonic acid units. The dialdehydes originated fromthe uronic acid units, and optionally from glucosamines, are preferably,and within a short time, further oxidized to dicarboxylates.

In Vitro Testing

Based on previous studies of Bisio et al. (Bisio A. et al. 2007,High-performance liquid chromatographic/mass spectrometric studies onthe susceptibility of heparin specie to cleavage by heparanase. SemThromb hemost 33 488-495), heparanase inhibiting activity was determinedin vitro by the group of Prof. Vlodaysky at the University of Haifa,Israel, according to the method described by Hammond et al. (Hammond etal. 2010, Development of a colorimetric assay for heparanase activitysuitable for kinetic analysis and inhibitor screening. Anal. Biochem.396, 112-6). Briefly, heparanase can cleave the syntheticpentasaccharide Fondaparinux, which is an antithrombotic drug,structurally corresponding to the antithrombin binding site of heparin.After heparanase cleavage, a trisaccharide and a reducing disaccharideare obtained. The latter can be easily quantified in order to assessheparanase activity. In the present examples, the assay solution (100ul) comprised 40 mM sodium acetate buffer pH 5.0 and 100 mM Fondaparinux(GlaxoSmithKline), with or without inhibitor sample. Heparanase wasadded to a final concentration of 140 pM to start the assay. The plateswere sealed with adhesive tape and incubated at 37° C. for 2-24 hours.The assay was stopped by addition of 100 μL of a solution of 1.69 mM4-[3-(4-iodophenyl)-1H-5 tetrazolio]-1,3-benzene disulfonate (WST-1,Aspep, Melbourne, Australia) in 0.11M NaOH. The plates were resealedwith adhesive tape and developed at 60° C. for 60 min. The absorbancewas measured at 584 nm (Fluostar, BMG, Labtech). In each plate, astandard curve constructed with D-galactose as the reducing sugarstandard was prepared in the same buffer and volume over the range of 2to 100 μM. The IC₅₀ value was determined. Because this assay has ahomogeneous substrate with a single point of cleavage, the kinetics andbiochemical parameters of the enzyme can be reliably characterized.Results obtained using the above described colorimetric assay werevalidated using sulfate labeled extracellular matrix (ECM) as substrate.Briefly, the ECM substrate is deposited by cultured corneal endothelialcells and hence closely resembles the subendothelial basement membranein its composition, biological function and barrier properties. Detailedinformation about the preparation of sulfate labeled ECM and its use forthe heparanase assay can be found in: Vlodaysky, I., Current Protocolsin Cell Biology, Chapter 10: Unit 10.4, 2001. The assay is highlysensitive, better resembles the in vivo conditions, but, due to itsbiological nature, it is semi-quantitative and limited in terms ofbiochemical parameters.

In Vivo Testing

The antimyeloma activity in vivo was tested substantially following theprocedure described in Yang Y et al. (Yang Y., et al. 2007, Thesyndecan-1 heparan sulfate proteoglycan is a viable target for myelomatherapy. Blood 110:2041-2048). Briefly, CB17 scid/scid mice aged 5 to 6weeks were obtained from Arlan (Indianapolis, Ind.) or Charles RiverLaboratories (USA). Mice were housed and monitored in the animalfacility of the University of Alabama at Birmingham. All experimentalprocedures and protocols were approved by the Institutional Animal Careand Use Committee. 1×10⁶ heparanase-expressing CAG myeloma cells (highor low expressing) were injected subcutaneously into the left flank ofeach mouse. Ten days after injection of tumor cells, mice were implantedwith Alzet osmotic pumps (Durect Corporation, Cupertino, Calif.) on theright flank. Pumps contained either solution of test compounds (newheparin derivatives) or PBS as control. The solution was deliveredcontinuously for 14 days. After 14 days, the animals were killed and thewet weight of the subcutaneous tumors and the mean sera kappa level wereassayed and compared among the experimental groups by log-rank test(p<0.05 was considered statistically significant).

Weekly luciferase bioluminescence imaging provides quantitative data onprimary tumors and tracks metastasis within bone as well as softtissues. Notably, the SCID-hu model is unique in that human tumor cellsare injected directly into small pieces of human fetal bone implantedsubcutaneously in SCID mice, thus closely recapitulating human myeloma.

General Procedure of NMR Analysis

Spectra were recorded at 25° C. on a Bruker Avance 500 spectrometer(Karlsruhe, Germany) equipped with a 5-mm TCI cryoprobe or with a 10 mmBBO probe. Integration of peak volumes in the spectra was made usingstandard Bruker TopSpin 2.0 software. The structure of dicarboxylateduronic acid residues was determined by two-dimensional heteronuclearexperiments that confirmed the presence of the tricarboxylate residuesand permitted the identification of their chemical shift. In the tablebelow the assigned chemical shifts of proton and carbons in position 1,4, 5 of the dicarboxylated residue, of glucosamine and 2-O-sulfatediduronic acid are reported.

Signals corresponding to residue position ¹H ppm ¹³C ppm CS-Uronic acidgsox 4.97 81.65 C4-Uronic acid gsox 4.59 81.22 Cl-Uronic acid gsox 5.01103.71 C4 3.63 79.14 Cl-Glucosamine NS (ANS) 5.40 99.17 Cl-ANS linked to(gsox) 5.03 100.19 Cl-Iduronic acid 2S 5.17 102.11

General Procedure for Calculation of Carboxyl Groups Increment

The increase of carboxyl groups in the uronic acid residues ofdicarboxylated heparin derivatives was calculated starting fromrespective values of molar ratio SO₃—/COO— of the starting material(unfractionated heparin or at least partially desulfated heparins andLMWHs) and of the dicarboxylated derivatives, evaluated byconductimetric titration (Casu B. and Gennaro U., 1975, Carbohydr Res39, 168-176). In particular, the sulfation degree of the startingmaterial is determined after the first oxidation step, on a reducedsample of the glycosaminoglycan oxidized intermediate (see examples4-7), while the sulfation degree of the final glycosaminoglycancarboxylated derivative is determined after the second oxidation step(see examples 8-11, 13-14).

SO₃ ⁻/CO₂ ⁻=A (ratio in starting materials) SO₃ ⁻=A/CO₂ ⁻SO₃ ⁻/CO₂ ⁻=B (ratio in dicarboxylated derivatives) SO₃ ⁻=B/CO₂ ⁻

Given that the number of sulfate groups does not change during the twosteps of oxidation, it can be concluded that the increase of thecarboxyl groups(CO₂-_((dicarboxylated derivatives))/CO₂-_((starting materials))) isequal to the ratio of the individual molar ratios A and B.

Carboxyl increment (C.I.)=AB=CO₂ ⁻ _((dicarboxylated derivatives))/CO₂ ⁻_((starting materials))⋅

Enzymatic Cleavage of Heparins and their Carboxylated Derivatives andHPLC/MS Analysis

The substrate (2-3 mg) was dissolved in a 1:1 (v/v) mixture of 100 mMsodium acetate buffer (pH 7.0) and 10 mM calcium acetate to obtain a 7.7mg/ml solution. To carry out the enzymatic cleavage 144 μI of themixture of 100 mM sodium acetate and 10 mM calcium acetate (1:1) and 3μI of heparinases mixture (heparin lyases I, II and III) (1 μI of eachlyase, 2 mU/μI enzyme solution) were added to 13 μI of the heparinsolution. The reaction mixture was stirred at 37° C. (Thermo-ShakerTS-100, Biosan) for 24 h. The reaction was stopped by adding 3 μI of 3%HCOOH. Each sample was diluted two times with water and analyzed byIPRP-HPLC/ESI-TOF (micrOTOF-Q, Bruker). A C18 kinetex and a gradient(0′-17% B, 15′-20% B, 55′-40% B, 100′ 50% B, 115′ 90% B) that use fase A(pH 6.25) and B (pH 7.95) with 10 mM DBA-CH₃COOH was used with a flowrate 100 μL/min.

Example 1 (G7669)

A sample of UFH (2.5 g of lot. G5842) in 1M NaOH (32 ml) was heated at60° C. for 30 min. After cooling at room temperature and neutralizationwith 2N HCl, the solution was dialyzed at room temperature for 3 daysagainst distilled water in membranes (cut-off: 3500 Da). Concentrationunder reduced pressure and freeze drying gave: 2.15 g (yield=80% w/w) ofan intermediate with 13% of the total uronic acid residues bearingepoxide group as determined by ¹³C-NMR. The sample was then dissolved inwater (32 ml) and kept under stirring at 70° C. for 2 days, in order tohydrolyze the epoxy groups. After concentration and freeze-drying, G7669was obtained.

Example 2 (G8661)

A sample of UFH (2.11 g of lot. G3378) in 27 ml of 1N NaOH was stirredat 60° C. for 30 min. Neutralization, cooling at room temperature anddialysis, concentration and freeze-drying (as described in Example 1)gave the intermediate G8637 (1.5 g). Since its ¹³C-NMR spectrumindicated the presence of epoxy groups, G8637 (1.5 g) was dissolved inwater (32 ml) and kept under stirring at 70° C. for 2 days, in order tohydrolyze the epoxy groups. After concentration and freeze-drying, G8661was obtained (1.5 g).

Example 3 (G8699)

A sample of UFH (2.01 g of lot. G3378) was processed as described inExample 2 to give the intermediate epoxy-containing derivative G8638. Asample of G8638 (1.4 g) was dissolved in water (32 ml) and heated understirring at 70° C. for 24 hours, to give, after concentration andfreeze-drying, 1.3 g of G8699.

Periodate Oxidation of Unfractionated Heparin and 2-O-DesulfatedDerivatives Example 4 (G7731)

Starting from sample G7669 of Example 1 (1.8 g, 13% 2-O-desulfation) inwater (52 ml), the solution was cooled at 4° C., stirred in the dark and52 ml of 0.2M NaIO₄ were added. After 16 hours, the excess of periodatewas quenched by adding ethylene glycol (5.2 ml) and after 1 hr. at 4° C.the reaction mixture was desalted by dialysis at 4° C. for 16 hours.After concentration under reduced pressure and freeze-drying, thedialdehyde-bearing G7731 was obtained (1.5 g), yield=83%. A smallportion of sample was reduced with NaBH₄ to measure MW=8,242 Da and SO₃⁻/COO⁻=2.46, by conductimetric titration.

Example 5 (G8425)

Starting from a sample of UFH (0.25 g, lot. G3378) and following thesame procedure described in Example 4, the dialdehyde-bearing G8425 wasobtained (0.24 g), yield=96% w/w.

Example 6 (G8678)

Starting from a sample of G8661 of Example 2 (1.5 g) and performing thesame procedure described in Example 4, the dialdehyde-bearing G8678 wasobtained (1.5 g). A small portion of sample was reduced with NaBH₄ tomeasure SO₃ ⁻/COO⁻=1.96 by conductimetric titration.

Example 7 (G8710)

Starting from a sample of G8699 of Example 3 (0.56 g) and following thesame procedure described in Example 4, the dialdehyde-bearing G8710 wasobtained (0.56 g). A small portion of sample was reduced with NaBH₄ tomeasure SO₃ ⁻/COO⁻=1.51 by conductimetric titration.

Oxidation of the Dialdehydic Uronic Acid Intermediates to theCorresponding Dicarboxylates Example 8 (G7927)

A sample of G7731 of Example 4 (0.3 g) was dissolved in water (29 ml),cooled at 0° C. in a double-necked round-bottomed flask and, understirring in nitrogen atmosphere, it was treated with an aqueous solution(6 ml) containing NaClO₂ (0.362 g). After a drop-wise addition ofglacial acetic acid (0.118 ml) up to reach pH 4.0, the reaction mixturewas stirred at room temperature for 24 hours. After further 3 hoursunder stirring at room temperature, by fluxing N₂, a colorless solutionwas obtained. The reaction mixture was neutralized with 0.5N NaOH anddesalted by dialysis as described in Example 4. Concentration andfreeze-drying gave G7927 (0.228 g), yield=76% w/w, having:

MW=6,450 Da;

SO₃ ⁻/COO⁻=1.23;Carboxyl increment (C.I.)=2In vitro heparanase inhibition analysis gave: IC₅₀=10 ng/ml.

Example 9 (G8437)

Starting from a sample of G8425 of Example 5 (0.25 g), in which only thenon-sulfated uronic acids naturally present in the chain of heparin(18%) were oxidized following the same procedure described in example 8,G8437 (0.21 g) was obtained by reducing to 1.5 hours the N₂ fluxingbefore neutralization of the reaction mixture, having:

MW=12,100 Da;

SO₃ ⁻/COO⁻=1.62;Carboxyl increment (C.I.): 1.43.

In vitro heparanase inhibition analysis gave: IC₅₀=10 ng/ml.

Example 10 (G8767)

Starting from samples of G8678 of Example 6 (1 g) and following theprocedure described in Example 8, G8767 (1.05 g) was obtained, having:

MW=8,800 Da;

SO₃ ⁻/COO⁻=1.27;Carboxyl increment (C.I.)=1.55.In vivo antimyeloma activity analysis (60 mg/kg day for 14 days) gave:52% tumor inhibition and 20% serum K inhibition.

Example 11 (G8733)

Starting from sample G8710 of Example 7 (0.56 g) and following the sameprocedure described in the Example 8, G8733 (0.453 g) was obtained,yield=80% w/w, having:

MW=5,540 Da;

SO₃ ⁻/COO—=0.97;Carboxyl increment (C.I.)=1.56.

In vivo antimyeloma activity analysis (60 mg/kg day for 14 days) gave:53% tumor inhibition.

Example 12 (G9685)

Starting from a sample of UFH (lot G3378) and following the proceduredescribed in Example 4, the dialdehyde derivative was obtained. Thedialdehyde derivative was further oxidized in the presence of sodiumchlorite, following the procedure of Example 8, obtaining thecarboxylated derivative G9685, MW=11,700 Da.

In vitro heparanase inhibition analysis gave: IC₅₀=10 ng/ml;

In vivo antimyeloma activity analysis (60 mg/kg day for 14 days) gave:68% tumor inhibition.

Example 13 (G7897)

A sample of G7731 of Example 4 (0.3 g) dissolved in water (3 ml) andtreated with 1.36 ml of NaClO₂ to pH 8.2-8.5 with HCl 4%. Then 1 mg ofTEMPO was added and pH was maintained a 7-7.5 with NaOH 1M for 24 hours.The reaction mixture was desalted by dialysis, concentrated andfreeze-dried to give G7897 (0.190 g), yield=63% w/w, having:

SO₃ ⁻/COO⁻=1.92;Carboxyl increment (C.I.)=1.28.

Example 14 (G9585)

Starting from a sample of 2-O-desulfated heparin (G9416, SO₃⁻/COO⁻=1.39), following the procedure described in Example 4, thedialdehyde derivative G9577 was obtained. A sample of G9577 (0.242 g)dissolved in water (21 ml) was cooled at 0° C. in a double-neckedround-bottomed flask and, under stirring in nitrogen atmosphere, it wastreated with an aqueous solution (1 ml) containing NaClO₂ (0.128 g).After a drop-wise addition of glacial acetic acid (0.084 ml) up to reachpH 4.0, the reaction mixture was stirred at room temperature for 24hours. After further 3 hours under stirring at room temperature, byfluxing N₂, a colorless solution was obtained. The reaction mixture wasneutralized with 0.5N NaOH and desalted by dialysis as described inExample 4. Concentration and freeze-drying gave G9585 (0.148 g),yield=61% w/w, having:

MW=4,700 Da;

SO₃ ⁻/COO⁻=0.82;Carboxyl increment (C.I.)=1.69.In vitro heparanase inhibition analysis gave: IC₅₀=44 ng/ml.

What is claimed is:
 1. A carboxylated derivative of a glycosaminoglycan,wherein the carboxylated derivative comprises the following structure:

wherein R₁ is SO₃ or Ac; R₂ is SO₃ or H.
 2. The carboxylated derivativeof a glycosaminoglycan according to claim 1, further comprising thefollowing two structures:

wherein R₂ is SO₃ or H; and R₃ is SO₃.
 3. The carboxylated derivative ofa glycosaminoglycan according to claim 1, wherein the glycosaminoglycanthat the carboxylated derivative is derived from is selected from one ofunfractionated heparin, low molecular weight heparin, and heparansulfate.
 4. The carboxylated derivative of a glycosaminoglycan accordingto claim 3, wherein the unfractionated heparin is 2-O-desulfatedunfractionated heparin, 2-N-desulfated unfractionated heparin or 2-O-,2-N-desulfated unfractionated heparin.
 5. The carboxylated derivative ofa glycosaminoglycan according to claim 3, wherein the low molecularweight heparin is 2-O-desulfated low molecular weight heparin,2-N-desulfated low molecular weight heparin or 2-O-, 2-N-desulfated lowmolecular weight heparin.
 6. The carboxylated derivative of aglycosaminoglycan according to claim 1, wherein the glycosaminoglycanthat the carboxylated derivative is derived from has a sulfation degree(SO₃ ⁻/COO⁻), determined by conductimetric titration as defined herein,of from 0.8 to 2.8.
 7. The carboxylated derivative of aglycosaminoglycan according to claim 6, wherein the glycosaminoglycanthat the carboxylated derivative is derived from has a sulfation degree(SO₃ ⁻/COO⁻), determined by conductimetric titration as defined herein,of from 0.9 to 2.5.
 8. The carboxylated derivative of aglycosaminoglycan according to claim 1, wherein the carboxylatedderivative of a glycosaminoglycan has a sulfation degree (SO₃ ⁻/COO⁻),determined by conductimetric titration as defined herein, of from 0.82to 1.92.
 9. The carboxylated derivative of a glycosaminoglycan accordingto claim 1, wherein said derivative has a molecular weight of from 8000to 30,000 Da.
 10. The carboxylated derivative of a glycosaminoglycanaccording to claim 1, wherein said derivative has a molecular weight offrom 3000 to 20000 Da.
 11. The carboxylated derivative of aglycosaminoglycan according to claim 1, wherein said derivative has amolecular weight selected of from 3500 to 12000 Da.
 12. A method oftreating an indication gaining benefit from inhibition of heparanase,comprising administering to a human in need of such treatment apharmaceutical composition comprising a therapeutically effective amountof the carboxylated derivative of the glycosaminoglycan of claim
 1. 13.A method of treating tumor metastasis or a tumor in a human, the methodcomprising administering to the human in need of such treatment apharmaceutical composition comprising a therapeutically effective amountof the carboxylated derivative of the glycosaminoglycan of claim
 1. 14.The method according to claim 13, wherein the tumor is myeloma.